This grant focuses on a relatively unexplored area, the bone marrow:bone interface. Its broad, long-term objectives are to systematically evaluate the influence of the bone marrow microenvironment on the development of human bone precursor (osteopoietic) cells. Many aspects of these studies are unique. Importantly, the proposal exams purified populations of primary bone marrow-derived human bone precursor cells, thus eliminating problems due to species specificities, or the use of cell lines. Further, these studies evaluate the responsiveness of these cells in chemically defined media, using recombinant or homogeneously purified growth factor and/or extracellular matrix (ECM) molecules to precisely define growth factor and ECM requirements. We propose to functionally and phenotypically characterize purified human bone precursor cell responsiveness to both early-and late-acting bone marrow growth factors, or combinations thereof. These studies will quantify cytokine effects on osteoprogenitor cells, and (pre)-osteoblast-like cell differentiation. Multi-parameter flow cytometry, and metabolic labeling/immunoprecipitation will be used to evaluate the expression, and production of various bone-related proteins. These studies also will use a highly-sensitive cell attachment assay to determine the interaction of human bone precursor cells to various bone marrow ECM molecules. Subsequently, the effect of bone marrow ECM molecules, alone or in combination with growth factors will be studied. The second goal of this proposal is to evaluate the actions of bone marrow stomal/accessory cells on human bone precursor cell development. These studies will address the phenotype of these accessory cells, and how they interact with osteopoietic cells to stimulate their development. As well RT-PCR will be used to determine the cytokines produced by these cells. Constitutive or induced cytokines will be evaluated for their affects on osteopoiesis. The final aim is to evaluate the ability of human bone precursor cells to elaborate plasminogen and/or plasminogen activator, and to determine whether plasmin-medicated ECM remodeling modulates osteopoiesis. Plasminogen and plasmin production will be monitored by Western and ELISA analysis. The involvement of plasmin in human bone precursor cells development is examined in studies using specific dominant/negative mutants of plasminogen. The above investigations thus use purified human bone precursor cells, recombinant bone marrow growth factors and ECM molecules in serum-free, chemically defined media to reduce a complex microenviroment to a series of testable variables. As such the data generated from these investigations will be important to understanding the relationship between the bone marrow microenvironment and bone cells in health and disease.